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Generation of monoclonal antibodies specific for mouse leukotriene B4 receptor 1 and N‐terminal FLAG sequence
Author(s) -
Sasaki Fumiyuki,
Saeki Kazuko,
Okuno Toshiaki,
Yokomizo Takehiko
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.551.12
BLT1 belongs to the G‐protein coupled receptor (GPCR) family, which is known as a chemoattractant receptor for leukotriene B 4 . To identify BLT1‐expressing cells by flowcytometry, we established a line of hybridoma cells (7A8) that produces a monoclonal antibody (mAb) for mouse BLT1 (mBLT1). We confirmed the specificity of 7A8 mAb using mBLT1‐overexpressing cells, and mouse leukocytes that endogenously express BLT1. The 7A8 mAb didn▪ft cross‐react with human BLT1, mouse BLT2 and other GPCRs. Biotin‐labeled 7A8 mAb stained wild‐type (WT) granulocytes (Gr1 hi ) and monocytes (Gr1 mid ), and this staining disappeared in BLT1 knockout (KO) granulocytes. BLT1 was also detected in F4/80, MHC class II or CD11b‐positive WT monocytes, and not in BLT1‐KO monocytes. In addition, we tried to deplete BLT1‐expressing leukocytes by i.p. injection of 7A8 mAb. After 24 h of injection, granulocyte and monocyte populations significantly decreased in peripheral blood, but lymphocyte population did not. In vitro differentiation/activation assay, BLT1 expression was induced during the differentiation into Th1 and Th17, and activated CD8 + T cells. Furthermore, we also generated a novel high‐affinity anti‐FLAG mAb (2H8), which is more sensitive and useful than commercially available antibodies (M2 and M5). Of note, 2H8 mAb detected with higher sensitivity for N‐terminal FLAG sequence of GPCRs.